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doi: 10.1242/10.1242/dev.00238

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Snail precedes Slug in the genetic cascade required for the specification and migration of the Xenopus neural crest

¹ Millennium Nucleus in Developmental Biology, Facultad de Ciencias, Universidad de Chile, Casilla 653, Santiago, Chile
² Instituto Cajal, CSIC, Doctor Arce 37, 28002, Madrid, Spain

View larger version (38K): [in a new window]   Fig. 1. Snail fusion proteins. The constructs used to produce the Snail fusion proteins are represented in this figure. The numbers above the bars indicate the amino acid number in the wild-type protein. Red is used for Xenopus Snail, green for Xenopus Slug, yellow for chick Snail and orange for chick Slug. N, amino terminal; ZnF, zinc-finger region; GR (light blue), glucocorticoid receptor domain; EnR (yellow), transcriptional repressor domain of the Engrailed protein of Drosophila; E1A (dark blue), transcriptional activator domain. See Materials and Methods for details.

View larger version (78K): [in a new window]   Fig. 2. Expression pattern of Snail and Slug analysed by whole-mount in situ hybridisation. (A) Dorsal view of a stage 9 embryo. An, animal pole; Veg, vegetal pole. Notice the expression in the dorsal marginal zone. (B) Dorsal view of a stage 11.5 embryo. b, dorsal blastopore lip. Snail is expressed in the prospective neural crest (arrowheads) and in a continuous band at the anterior border of the neural plate (arrow). (C) Same embryo as in B (stage 11.5) but hybridised for Slug expression. No expression is seen in the ectoderm (white arrowheads). (D) Dorsovegetal view of the embryo shown in B. Arrow indicates Snail staining in the anterior ectoderm. This is also visible around the blastopore lip in the marginal zone, apart from the dorsal region where Snail has been switched off. (E) Dorsal view of a stage 12.5 embryo. Expression is visible in the mesoderm near the blastopore and in the prospective neural crest (arrowhead). An, animal pole; Veg, vegetal pole. (F) Earliest Slug expression. Dorsal view of a stage 12.5 embryo. Expression in the prospective neural crest (arrowheads). An, animal pole; Veg, vegetal pole. (G) Anterior view of a stage 17 embryo. Expression in the superficial (white arrowheads) and in the deep (black arrowheads) layers of the ectoderm and in the anterior neural fold (a, arrow). (H,I) Schematic representation of Snail and Slug expression at stages 11 and 12.5. Anterior is upwards and posterior is downwards.

View larger version (79K): [in a new window]   Fig. 3. Snail participates in the early specification of the neural crest. One blastomere of a two-cell stage embryo was injected with 700 pg of Xsnail-GR mRNA, treated with dexamethasone at stage 12.5, fixed at stage 19, and the expression of several genes analysed. The arrowheads indicate the injected side that contained FLDx (see Materials and Methods). Anterior is towards the left. (A-D) Neural crest markers. Notice the expansion of the markers on the injected side. (A) Snail expression. (B) Slug expression. (C) Twist expression. (D) FoxD3 expression. (E) Expression of the neural plate marker Sox2, is reduced on the injected side. The broken line indicates the dorsal midline and the brackets indicate the width of the neural plate. (F) Expression of the epidermal marker Cytokeratin 81A (dorsal view), is almost completely inhibited on the injected side. (G,H) Lateral views of the same embryo where the inhibition of Cytokeratin expression is better assessed. G corresponds to the injected side.

View larger version (99K): [in a new window]   Fig. 4. The expansion of the neural crest territory induced by Snail or Slug does not require cell proliferation. One blastomere of a two-cell stage embryo was injected with 700 pg of Xsnail-GR mRNA (A,B) or Xslug-GR (C,D), treated with dexamethasone and HUA at stage 12.5, fixed at stage 19 and the expression of the neural crest markers Snail (A,C) and Slug (B,D) analysed. The injected side, which can be recognised by the blue FLDx staining, is indicated by the arrowhead. Note the expansion in the expression of the neural crest markers on the injected side. (E) HUA treated and (F) control embryos stained for histone H3 to verify the blockade in cell proliferation induced by the treatment. The inset shows a higher magnification of the embryos. Note the staining in absence of HUA treatment, but the lack of staining after HUA treatment.

View larger version (29K): [in a new window]   Fig. 5. Snail is able to activate the expression of neural crest markers and block BMP4 expression. Embryos were injected at the one-cell stage with 1 ng of Snail mRNA. At stage 8, the animal caps were dissected and cultured until the equivalent of stage 20, when mRNA was isolated and assayed by RT-PCR as described in the Materials and Methods. Embryo: whole embryo showing the expression of all the markers. H4 was used as a loading control. Control: uninjected animal caps. Xsnail 1 ng: animal caps taken from embryos injected with Snail mRNA. (A) Note that Snail is able to induce early and late neural crest markers in the absence of neural plate and mesodermal markers. (B) Note the inhibition in the expression of BMP4 after Snail mRNA injection.

View larger version (54K): [in a new window]   Fig. 6. Inhibition of Snail activity blocks the expression of neural crest markers. One blastomere of a 2-cell stage embryo was injected with 700 pg of the different dominant negative constructs, treated with dexamethasone at stage 12.5, fixed at stage 19, and the expression of the neural crest markers analysed. The injected side is indicated by an arrowhead. (A-D) XsnailZnFGR: dominant-negative of the Snail zinc fingers. Note that the dominant negative construct inhibited the expression of all the neural crest markers analysed. (E,F) Rescue of XsnailZnFGR by XsnailGR: both mRNAs were injected in equivalent amounts and analysed as previously described. Note the normal expression of the neural crest markers in the injected side. (G,H) SnailN-GR: dominant-negative using the Snail N-terminal domain. Note that the dominant-negative constructs inhibited the expression of all the neural crest markers analysed. (I,J) Rescue of XsnailNGR by XsnailGR: both mRNAs were injected in equivalent amounts and analysed as previously described. Note the normal expression of the neural crest markers in the injected side.

View larger version (68K): [in a new window]   Fig. 7. Snail lies upstream of Slug in the cascade leading to neural crest development. One blastomere of a two-cell stage embryo was co-injected with the different dominant-negative constructs and the wild type, treated with dexamethasone at stage 12.5, fixed at stage 19 and the expression of the neural crest markers analysed. The injected side is indicated by an arrowhead. (A,B) XsnailZnFGR rescued by XslugGR: the effect of the zinc fingers dominant-negative Snail construct was rescued by co-expression of Slug. Note the normal expression of neural crest markers in the injected side. (C,D) Effect of injecting XslugNGR dominant-negative construct. Note the inhibition in the expression of the markers in the injected side. (E,F) Co-injection of XslugNGR and XsnailGR: note that the effect of the dominant-negative Slug construct can not be rescued by co-expression of Snail.

View larger version (65K): [in a new window]   Fig. 8. Snail functions as a transcriptional repressor. One blastomere of a two-cell stage embryo was injected with 700 pg of a Snail repressor construct (A-D) or the Snail activator construct (E-H), treated with dexamethasone at stage 12.5, fixed at stage 19, and the expression of neural crest markers analysed. Arrowhead, injected side. Note that the Snail repressor construct (XsnailZnF-GR-EnR) produced an expansion of the neural crest markers on the injected side (A-D), while the Snail activator lead to an inhibition in the expression of the markers (E-H).

View larger version (62K): [in a new window]   Fig. 9. Snail controls neural crest migration. One blastomere of a two-cell stage embryo was injected with 700 pg of SnailGR (A,D), its dominant negative (B,E) or SlugGR (C,F) treated with dexamethasone at stage 16, fixed between stages 22 and 23, and the expression of the neural crest markers Slug analysed. (A-C) Injected side (arrowhead). (D-F) Uninjected side of the embryos shown in A-C. The leading edge of migration is indicated with a broken line. Note that SnailGR (A,D) and SlugGR (C,F) produces a stronger migration in the injected side; while the injection of ZnFXsnailGR (B,E) leads to an inhibition in the migration of the crest cells.

View larger version (95K): [in a new window]   Fig. 10. Functional equivalence of the Snail genes assayed in Xenopus embryos. One blastomere of a two-cell stage embryo was injected with 500 pg of mRNA encoding for different members of the Snail gene family, treated with dexamethasone at stage 12.5, fixed at stage 25, and the expression of the neural crest marker Slug analysed. Arrowhead indicates injected side. (A,B) Xenopus genes: Xsnail-GR (A) or Xslug-GR (B). (C,D) Chick genes: Snail-GR (C) or Slug-GR (D). Note that in all the injected sides of the embryos a more vigorous and larger population of migratory crest cells (asterisks).